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Abstract:

The invention provides compositions and methods relating to the elevation
of glucoraphanin compared to standard Brassica oleracea varieties. The
invention also relates to the production of hybrid varieties having
desired glucosinolate contents. The invention further provides plants,
plant parts, and seeds comprising such traits and comprising a Myb28
allele from Brassica villosa that is not genetically linked to an ELONG
allele from Brassica villosa.

Claims:

1. A Brassica oleracea plant comprising a Myb28 allele from Brassica
villosa and lacking an ELONG allele from Brassica villosa genetically
linked to said Myb28 allele, wherein the Myb28 allele confers elevated
glucosinolates when compared to a plant that lacks the Myb28 allele.

2. The plant of claim 1, wherein the plant is a broccoli plant.

3. The plant of claim 1, wherein the plant is inbred.

4. The plant of claim 1, wherein the plant is hybrid.

5. The plant of claim 1, wherein the plant is homozygous for said Myb28
allele from Brassica villosa.

6. The plant of claim 1, wherein the plant is heterozygous for said Myb28
allele from Brassica villosa.

7. The plant of claim 1, wherein the ELONG allele is from Brassica
oleracea.

8. A plant part of the plant of claim 1.

9. The plant part of claim 8, wherein the part is a leaf, a ovule, a
floret, pollen, a head, or a cell.

10. A seed that produces the plant of claim 1.

11. A Brassica oleracea plant comprising a chromosomal segment that
comprises a Myb28 allele from Brassica villosa and lacking an ELONG
allele from Brassica villosa genetically linked to said Myb28 allele,
wherein the segment confers elevated glucosinolates relative to a plant
lacking the Myb28 allele, wherein a sample of seed comprising the
chromosomal segment was deposited under ATCC Accession Number PTA-13165.

12. A seed that produces the plant of claim 11.

13. A plant part of the plant of claim 11.

14. The plant part of claim 13, wherein the part is a leaf, an ovule, a
floret, pollen, a head, or a cell.

15. A recombined DNA segment comprising a Myb28 allele from Brassica
villosa and an ELONG allele from Brassica oleracea.

16. The DNA segment of claim 15, further defined as comprised within a
cell.

17. The DNA segment of claim 15, further defined as comprised within a
seed.

18. The DNA segment of claim 15, further defined as comprised within a
plant.

19. A method for obtaining a Brassica plant comprising a desired
glucosinolate composition comprising: (a) obtaining a Brassica plant
heterozygous for a Myb28 allele from Brassica villosa that confers
elevated glucosinolates and is genetically linked in the plant to an
ELONG allele from Brassica villosa; (b) obtaining progeny of the plant;
and (c) selecting at least a first progeny plant in which recombination
has occurred such that the progeny comprises the Myb28 allele but not the
ELONG allele from Brassica villosa, wherein the progeny plant possesses a
desired glucosinolate composition as a result of the presence of the
Myb28 allele but not the ELONG allele from Brassica villosa.

20. The method of claim 19, wherein selecting the progeny plant comprises
identifying a progeny plant that (1) comprises a genetic marker
genetically linked to the Myb28 allele in Brassica villosa and/or lacks a
genetic marker present at the corresponding locus in said Brassica plant,
and (2) lacks a genetic marker genetically linked to the ELONG allele
from Brassica villosa and/or comprises a genetic marker present at the
corresponding locus from said Brassica plant.

21. The method of claim 20, wherein selecting a progeny plant comprises
detecting a polymorphism that is found in the genome of said plant
flanked by the complements of SEQ ID NO:1 and SEQ ID NO:2.

22. The method of claim 21, wherein (a) the allele(s) are detected by a
PCR-based method using oligonucleotide primer pair(s).

23. The method of claim 20, wherein selecting a progeny plant comprises
detecting a polymorphism in said progeny plant that is shown in FIG. 5.

25. A plant produced by the method of claim 19 or a progeny thereof
comprising said Myb28 allele but not the ELONG allele from Brassica
villosa.

26. A part of the plant of claim 25 selected from the group consisting of
a cell, a seed, a root, a stem, a leaf, a head, a flower, and pollen.

27. A method for producing a hybrid Brassica oleracea plant with elevated
glucosinolate content comprising crossing a first Brassica oleracea
parent plant with a second Brassica oleracea plant of a different
genotype, wherein the first parent plant comprises a Myb28 allele from
Brassica villosa that lacks an ELONG allele from Brassica villosa
genetically linked to said Myb28 allele, wherein the Myb28 allele confers
elevated glucosinolates relative to a plant lacking the Myb28 allele.

28. The method of claim 27, further comprising producing a plurality of
hybrid Brassica oleracea plants comprising crossing the first Brassica
oleracea parent plant with a plurality of second Brassica oleracea plants
of different genotypes.

29. A method of producing a Brassica oleracea plant with a desired
elevated glucosinolate content comprising introgressing into the plant a
chromosomal segment comprising a Myb28 allele from Brassica villosa and
lacking an ELONG allele from Brassica villosa genetically linked to said
Myb28 allele, wherein segment confers a desired glucosinolate content
relative to a plant lacking the segment, wherein a sample of seed
comprising the chromosomal segment is deposited under ATCC Accession No.
PTA-13165.

[0002] The sequence listing that is contained in the file named
"SEMB008US_ST25.txt", which is 8 kilobytes as measured in Microsoft
Windows operating system and was created on Aug. 28, 2013, is filed
electronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to the development and use of
Brassica oleracea plants with a recombined chromosomal segment.

[0005] 2. Description of Related Art

[0006] Glucosinolates are allelochemicals present in 16 families of plant
species, especially in Brassicaceae, of which broccoli is a notable
example. Although there are over 120 different glucosinolates identified
in nature, closely related taxonomic groups typically contain only a
small number of such compounds. Glucosinolates that are dominant in
broccoli such as glucoraphanin and glucoiberin are derived biochemically
from the amino acid methionine. In the glucosinolate pathway, methionine
is converted to homo-methionine and dihomomethionine by the activity of
the ELONG (elongation) locus by adding a single carbon unit to the tail
each time. Homo-methionine is eventually converted to 3-methylthiopropyl
glucosinolate (glucoiberin; "MSP") while dihomo-methionine is converted
to 4-methylthiobutyl glucosinolate (glucoraphanin; "MSB"). These
glucosinolates (glucoiberin and glucoraphanin) are potent inducers of
phase II detoxification enzymes, such as glutathione-S-transferase and
quinone reductase, which promote the metabolism and excretion of
potential carcinogens.

SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides a Brassica oleracea
plant comprising a Myb28 allele from Brassica villosa and lacking an
ELONG allele from Brassica villosa genetically linked to said Myb28
allele, wherein the Myb28 allele confers elevated glucosinolates when
compared to a plant that lacks the Myb28 allele.

[0008] In one embodiment, the plant is a broccoli plant. In other
embodiments, the plant is inbred or hybrid. In another embodiment, the
plant is homozygous for said Myb28 allele from Brassica villosa. In yet
another embodiment, the plant is heterozygous for said Myb28 allele from
Brassica villosa. In still another embodiment, the ELONG allele is from
Brassica oleracea.

[0009] In another aspect, the present invention provides a part of a plant
of the invention. In some embodiments, the plant part may further be
defined as a leaf, an ovule, a floret, pollen, a head, or a cell.

[0010] In yet another aspect, the present invention provides a seed that
produces a plant of the invention.

[0011] In still another aspect, the present invention provides a Brassica
oleracea plant comprising a chromosomal segment that comprises a Myb28
allele from Brassica villosa and lacking an ELONG allele from Brassica
villosa genetically linked to said Myb28 allele, wherein the segment
confers elevated glucosinolates relative to a plant lacking the Myb28
allele, and wherein a sample of seed comprising the chromosomal segment
was deposited under ATCC Accession Number PTA-13165. In one embodiment,
the invention provides a seed that produces such a plant. In another
embodiment, the invention provides a plant part, wherein the part is a
leaf, an ovule, a floret, pollen, a head, or a cell. In another
embodiment the plant is a B. oleracea plant.

[0012] In another aspect of the present invention a recombined DNA segment
comprising a Myb28 allele from Brassica villosa and an ELONG allele from
Brassica oleracea is provided. In one embodiment, the DNA segment is
further defined as comprised within a cell. In another embodiment, the
DNA segment is further defined as comprised within a seed. In yet another
embodiment, the DNA segment is further defined as comprised within a
plant.

[0013] In another aspect, the present invention provides a method for
obtaining a Brassica plant comprising a desired glucosinolate composition
comprising: a) obtaining a Brassica plant heterozygous for a Myb28 allele
from Brassica villosa that confers elevated glucosinolates and is
genetically linked in the plant to an ELONG allele from Brassica villosa;
(b) obtaining progeny of the plant; and (c) selecting at least a first
progeny plant in which recombination has occurred such that the progeny
comprises the Myb28 allele but not the ELONG allele from Brassica
villosa, wherein the progeny plant possesses a desired glucosinolate
composition as a result of the presence of the Myb28 allele but not the
ELONG allele from Brassica villosa.

[0014] In one embodiment, selection of the progeny plant comprises
identifying a progeny plant that (1) comprises a genetic marker
genetically linked to the Myb28 allele in Brassica villosa and/or lacks a
genetic marker present at the corresponding locus in said Brassica plant,
and (2) lacks a genetic marker genetically linked to the ELONG allele
from Brassica villosa and/or comprises a genetic marker present at the
corresponding locus from said Brassica plant.

[0015] In another embodiment, selection of the progeny plant comprises
detecting a polymorphism that is found in the genome of said plant
flanked by the complements of SEQ ID NO:1 and SEQ ID NO:2. In a further
embodiment, such allele(s) are detected by a PCR-based method using
oligonucleotide primer pair(s). In another embodiment, selection of the
progeny plant comprises detecting a polymorphism in said progeny plant
that is shown in FIG. 5. In a further embodiment, the Brassica plant may
be a B. oleracea plant.

[0016] In yet a further aspect, the invention provides a plant produced by
a method of the invention or a progeny thereof comprising the Myb28
allele but not the ELONG allele from Brassica villosa. In one embodiment,
the invention provides a part of such a plant. In another embodiment, the
part of the plant is selected from the group consisting of a cell, a
seed, a root, a stem, a leaf, a head, a flower, and pollen.

[0017] In another aspect, the invention provides a method for producing a
hybrid Brassica oleracea plant with elevated glucosinolate content
comprising crossing a first Brassica oleracea parent plant with a second
Brassica oleracea plant of a different genotype, wherein the first parent
plant comprises a Myb28 allele from Brassica villosa that lacks an ELONG
allele from Brassica villosa genetically linked to said Myb28 allele,
wherein the Myb28 allele confers elevated glucosinolates relative to a
plant lacking the Myb28 allele. In one embodiment, the method further
comprises producing a plurality of hybrid Brassica oleracea plants
comprising crossing the first Brassica oleracea parent plant with a
plurality of second Brassica oleracea plants of different genotypes.

[0018] In still another aspect, the invention provides a method of
producing a Brassica oleracea plant with a desired elevated glucosinolate
content comprising introgressing into the plant a chromosomal segment
comprising a Myb28 allele from Brassica villosa and lacking an ELONG
allele from Brassica villosa genetically linked to said Myb28 allele,
wherein segment confers a desired glucosinolate content relative to a
plant lacking the segment, wherein a sample of seed comprising the
chromosomal segment is deposited under ATCC Accession No. PTA-13165.

[0019] The term "about" is used to indicate that a value includes the
standard deviation of error for the device or method being employed to
determine the value. The use of the term "or" in the claims is used to
mean "and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and to "and/or."
When used in conjunction with the word "comprising" or other open
language in the claims, the words "a" and "an" denote "one or more,"
unless specifically noted. The terms "comprise," "have" and "include" are
open-ended linking verbs. Any forms or tenses of one or more of these
verbs, such as "comprises," "comprising," "has," "having," "includes" and
"including," are also open-ended. For example, any method that
"comprises," "has" or "includes" one or more steps is not limited to
possessing only those one or more steps and also covers other unlisted
steps. Similarly, any plant that "comprises," "has" or "includes" one or
more traits is not limited to possessing only those one or more traits
and covers other unlisted traits.

[0020] Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It should
be understood, however, that the detailed description and any specific
examples provided, while indicating specific embodiments of the
invention, are given by way of illustration only, since various changes
and modifications within the spirit and scope of the invention will
become apparent to those skilled in the art from this detailed
description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 depicts the head from hybrid broccoli varieties Ironman
(left) and RX05991199 (right). Varieties were grown in Summer at a
spacing of 50×50 cm.

[0022] FIG. 2 shows a horizontal cross section of stems from hybrid
broccoli varieties Ironman (left) and RX05991199 (right). Varieties were
grown in Autumn at a spacing of 50×50 cm.

[0023] FIG. 3 shows a profile of the head and stem from hybrid broccoli
varieties Ironman (left) and RX05991199 (right). Varieties were grown in
Autumn at a spacing of 50×50 cm.

[0024] FIG. 4 shows assay results for different ELONG alleles with the
QTL1-BoGLS-ELONG marker. The V allele is the B. villosa allele associated
with Myb28, which results in high 3-MSP/4-MSB ratio and high total
glucosinolates. The A, B and C alleles are examples of alleles found in
broccoli without a B. villosa ELONG allele.

[0025] FIG. 5 shows an alignment between a consensus sequence of the Myb28
locus from B. villosa contained in broccoli variety FT69, and a consensus
sequence of the corresponding locus from broccoli without increased level
of glucosinolate, e.g. B. oleracea, (Oleracea) (SEQ ID NOs.:8-9).

DETAILED DESCRIPTION OF THE INVENTION

[0026] The invention provides methods and compositions relating to plants,
seeds and derivatives of Brassica oleracea plants comprising a new
recombined introgression from Brassica villosa capable of conferring
elevated 4-methylsulfinylbutyl glucosinolates (MSB), also known as
glucoraphanin. It was also surprisingly found that plants comprising the
introgression were capable of consistently producing hybrids that
exhibited elevated glucoraphanin content relative to glucoiberin, while
results were substantially more variable when using a parent line
comprising the non-recombined introgression, such as in the case of the
Myb28 donor parent of the hybrid PS05151639, which comprises Myb28 and
ELONG alleles from Brassica villosa (US Patent Appln Pub No.
2011/0055945). The ability to produce multiple elite hybrid progeny with
elevated glucoraphanin content and/or ratio of glucoraphanin to
glucoiberin from a single inbred parent is significant in that the number
of elite Brassica oleracea parent lines available to produce hybrid
varieties is limited. The reduced introgression therefore substantially
increases the utility of a given inbred and allows, for example, the
production of multiple hybrids potentially adapted to different growing
environments, end uses, or other criteria, each having a desired
glucoraphanin content.

[0027] The new "reduced introgression" comprising a Myb28 locus from
Brassica villosa and lacking the ELONG locus of Brassica villosa that has
to date been genetically linked thereto is capable of consistently
conferring in hybrids derived from a plant comprising the introgression
elevated glucoraphanin relative to glucoiberin. One aspect of the current
invention thus concerns methods for obtaining a Brassica oleracea plant
comprising at least one such reduced introgression, wherein the resulting
Brassica oleracea plant and/or progeny derived therefrom displays a
desired glucoraphanin content relative to a control plant lacking the
introgression. The invention thus provides plants possessing a desired
glucoraphanin content conferred by a reduced introgression of the
invention. In certain embodiments, methods for obtaining such plants
comprise obtaining a Brassica oleracea plant heterozygous for a Myb28
allele from Brassica villosa that confers elevated glucosinolates and is
genetically linked in the plant to an ELONG allele from Brassica villosa,
obtaining progeny from such a plant, and selecting one or more such
progeny plants wherein genetic recombination has occurred such that the
progeny comprises an Myb28 allele from Brassica villosa, but does not
comprise an ELONG allele from Brassica villosa. Such progeny or further
progeny thereof may also possess a desired glucoraphanin content as a
result of the presence of the Myb28 allele but not the ELONG allele from
Brassica villosa. In particular embodiments, the method may comprise
obtaining a progeny plant that comprises such allele(s) by identifying
one or more genetic markers genetically linked to the Myb28 and/or ELONG
allele(s). Identifying the genetic markers may comprise a phenotypic, a
genetic, or a biochemical test, and may include screening a parent and/or
progeny plant for the presence of, for instance, one or more allele
described herein, including, for example, a Myb28 allele from B. villosa,
an ELONG allele from B. villosa and an ELONG allele from B. oleracea.

[0028] Certain traits such as a glucoraphanin content relative to
glucoiberin content, or the unpredictability of the type of
glucosinolates in hybrid progeny heterozygous for a Brassica villosa
Myb28 allele, were found to co-locate with a glucosinolate trait in the
Myb28 allele from Brassica villosa and ELONG allele from Brassica villosa
introgressions. Thus, formation of a "reduced" introgression is
understood to be caused by recombination event(s) in the vicinity of the
Myb28 and ELONG QTL(s). Lines comprising a reduced introgression, i.e.,
which have undergone a recombination event close to the QTL having
elevated glucosinolates may efficiently be screened by use of molecular
and/or phenotypic markers. Thus, plant populations or progeny of such
populations, segregating (i.e., heterozygous) with respect to the QTL
specified by Myb28 and ELONG introgressions, may be screened for plants
having a recombinant phenotype, e.g. elevated glucoraphanin levels
relative to glucoiberin levels.

[0029] In other embodiments, a method of the invention may comprise
identifying a Brassica oleracea plant comprising a Brassica
villosa-derived reduced introgression, and comprising a meiotic
recombination between Myb28 and ELONG alleles as described herein. In
particular embodiments, identifying the introgression may comprise
measuring glucoraphanin and/or glucoiberin using standard protocols. In
certain embodiments, a plant of the invention comprising a reduced
introgression as disclosed herein comprises an elevated average
proportion of glucoraphanin relative to glucoiberin compared to a plant
comprising Myb28 and ELONG alleles from Brassica villosa, or a plant
lacking a Myb28 allele from Brassica villosa. In one embodiment, such a
plant comprising an elevated average proportion of glucoraphanin relative
to glucoiberin is an inbred line, and in another embodiment is defined as
a F1 hybrid having as one or more parent a plant comprising a reduced
introgression of the invention. In particular embodiments, a plant of the
invention is provided comprising a ratio of glucoraphanin to glucoiberin
of about 10:1, 12:1, 15:1, 18:1, 20:1, 23:1, 25:1, 28:1, 30:1, 35:1 and
about 40:1. In one aspect, an increase in glucoraphanin content may be
calculated in reference to a standard Brassica oleracea variety, such as
the broccoli variety Ironman.

[0030] One aspect of the current invention concerns methods for crossing a
plant comprising a reduced Myb28/ELONG introgression provided herein with
itself or a second plant and the seeds and plants produced by such
methods. These methods can be used for production and propagation of
cultivated Brassica oleracea plants displaying desired glucosinolate
compositions, including MSB and/or MSP content. Yet further, the plants
of the current invention having elevated glucosinolate content comprise
improved nutritional value of the plant relative to plants without
elevated glucosinolates. The methods also can be used to produce hybrid
Brassica oleracea seeds and the plants grown therefrom. Hybrid seeds are
produced by crossing such lines with a second Brassica oleracea parent
line. The hybrids may be heterozygous or homozygous for the reduced
introgression.

[0031] Brassica villosa is a wild species endemic to northwest and central
Sicily, and thus a Myb28 allele could be obtained by one of skill in the
art from a plant selected from the wild. Alternatively, Myb28 alleles are
known in the art and may be obtained from other sources for use with the
invention, including SNR 347 (FT69; referred to as 428-11-69 in Mithen et
al., Theor Appl Genet, 106:727-734; 2003), BR384-014, SNP13 (580333),
SNP88 (BRM51-1210), BR384-020, B1639 (ATCC Accession Number PTA-9676),
BRM51-1162 (ATCC Accession Number PTA-9675) and RX 05991199 (ATCC
Accession No. PTA-13165). In accordance with the invention, a plant
provided herein will generally lack an ELONG allele from Brassica villosa
genetically linked to the Myb28 allele. This can be achieved according to
the invention through crossing a plant comprising Myb28 and ELONG alleles
from Brassica villosa with Brassica plants not comprising the Myb28 and
ELONG alleles from Brassica villosa, including standard Brassica oleracea
varieties. This includes the many broccoli varieties well known in the
art, among others.

[0032] The goal of vegetable breeding is to combine various desirable
traits in a single variety/hybrid. Such desirable traits may include any
trait deemed beneficial by a grower and/or consumer, including greater
yield, resistance to insects or disease, tolerance to environmental
stress, and nutritional value. Breeding techniques used in an attempt to
obtain desired traits take advantage of a plant's method of pollination.
There are two general methods of pollination: a plant self-pollinates if
pollen from one flower is transferred to the same or another flower of
the same plant. A plant cross-pollinates if pollen comes to it from a
flower of a different plant.

[0033] The development of uniform varieties requires the development of
homozygous inbred plants, the crossing of these inbred plants, and the
evaluation of the crosses. Pedigree breeding and recurrent selection are
examples of breeding methods that have been used to develop inbred plants
from breeding populations. Those breeding methods combine the genetic
backgrounds from two or more plants or various other broad-based sources
into breeding pools from which new lines and hybrids derived therefrom
are developed by selfing and selection of desired phenotypes.

[0034] In accordance with the invention, novel varieties may be created by
crossing plants of the invention followed by generations of selection as
desired and inbreeding for development of uniform lines. New varieties
may also be created by crossing with any second plant. In selecting such
a second plant to cross for the purpose of developing novel lines, it may
be desired to choose those plants which either themselves exhibit one or
more selected desirable characteristics or which exhibit the desired
characteristic(s) when in hybrid combination. Once initial crosses have
been made, inbreeding and selection take place to produce new varieties.
For development of a uniform line, often five or more generations of
selfing and selection are typically involved.

[0035] Uniform lines of new varieties may also be developed by way of
doubled-haploids. This technique allows the creation of true breeding
lines without the need for multiple generations of selfing and selection.
In this manner true breeding lines can be produced in as little as one
generation. Haploid embryos may be produced from microspores, pollen,
anther cultures, or ovary cultures. The haploid embryos may then be
doubled autonomously, or by chemical treatments (e.g. colchicine
treatment). Alternatively, haploid embryos may be grown into haploid
plants and treated to induce chromosome doubling. In either case, fertile
homozygous plants are obtained. In accordance with the invention, any of
such techniques may be used in connection with a plant of the present
invention and progeny thereof to achieve a homozygous line.

[0036] Backcrossing can also be used to improve an inbred plant.
Backcrossing transfers a specific desirable trait, such as elevated
glucoraphanin, from one inbred or non-inbred source to a variety that
lacks that trait. This can be accomplished, for example, by first
crossing a parent (A) (recurrent parent) to a donor inbred (non-recurrent
parent), which carries the appropriate locus or loci for the trait in
question. The progeny of this cross are then mated back to the recurrent
parent (A) followed by selection in the resultant progeny for the desired
trait to be transferred from the non-recurrent parent. After five or more
backcross generations with selection for the desired trait, the progeny
are heterozygous for loci controlling the characteristic being
transferred, but are like the first parent for most or almost all other
loci. The last backcross generation would be selfed to give pure breeding
progeny for the trait being transferred.

[0037] The selection of a suitable recurrent parent is an important step
for a successful backcrossing procedure. The goal of a backcross protocol
is to alter or substitute a single trait or characteristic in the
original variety. To accomplish this, a single locus of the recurrent
variety is modified or substituted with the desired locus from the
nonrecurrent parent, while retaining essentially all of the rest of the
desired genetic, and therefore the desired physiological and
morphological constitution of the original variety. The choice of the
particular nonrecurrent parent will depend on the purpose of the
backcross; one of the major purposes is to add some commercially
desirable trait to the plant. The exact backcrossing protocol will depend
on the characteristic or trait being altered to determine an appropriate
testing protocol. Although backcrossing methods are simplified when the
characteristic being transferred is a dominant allele, a recessive allele
may also be transferred. It may be necessary to introduce a test of the
progeny to determine if the desired characteristic has been successfully
transferred.

[0038] Brassica oleracea varieties can also be developed from more than
two parents. The technique, known as modified backcrossing, uses
different recurrent parents during the backcrossing. Modified
backcrossing may be used to replace the original recurrent parent with a
variety having certain more desirable characteristics or multiple parents
may be used to obtain different desirable characteristics from each.

[0039] Many single locus traits have been identified that are not
regularly selected for in the development of a new inbred but that can be
improved by backcrossing techniques. Single locus traits may or may not
be transgenic; examples of these traits include, but are not limited to,
male sterility, herbicide resistance, resistance to bacterial, fungal, or
viral disease, insect resistance, restoration of male fertility, modified
fatty acid or carbohydrate metabolism, and enhanced nutritional quality.
These comprise genes generally inherited through the nucleus.

[0040] Direct selection may be applied where the single locus acts as a
dominant trait. Selection of Brassica plants for breeding is not
necessarily dependent on the phenotype of a plant and instead can be
based on genetic investigations. For example, one can utilize a suitable
genetic marker which is closely genetically linked to a trait of
interest. One of these markers can be used to identify the presence or
absence of a trait in the offspring of a particular cross, and can be
used in selection of progeny for continued breeding. This technique is
commonly referred to as marker assisted selection. Any other type of
genetic marker or other assay which is able to identify the relative
presence or absence of a trait of interest in a plant can also be useful
for breeding purposes.

B. PLANTS DERIVED FROM A PLANT OF THE INVENTION BY GENETIC ENGINEERING

[0042] Many useful traits that can be introduced by backcrossing, as well
as directly into a plant, are those, which may be introduced by genetic
transformation techniques. Genetic transformation may therefore be used
to insert a selected transgene into a Brassica plant of the invention or
may, alternatively, be used for the preparation of transgenes, which can
be introduced by backcrossing. Methods for the transformation of plants,
including Brassica, are well known to those of skill in the art.

[0043] Vectors used for the transformation of plant cells are not limited
so long as the vector can express an inserted DNA in the cells. For
example, vectors comprising promoters for constitutive gene expression in
Brassica cells (e.g., cauliflower mosaic virus 35S promoter) and
promoters inducible by exogenous stimuli can be used. Examples of
suitable vectors include pBI binary vector. The "Brassica cell" into
which the vector is to be introduced includes various forms of Brassica
cells, such as cultured cell suspensions, protoplasts, leaf sections, and
callus.

[0044] A vector can be introduced into Brassica cells by known methods,
such as the polyethylene glycol method, polycation method,
electroporation, Agrobacterium-mediated transfer, particle bombardment
and direct DNA uptake by protoplasts.

[0045] To effect transformation by electroporation, one may employ either
friable tissues, such as a suspension culture of cells or embryogenic
callus or alternatively one may transform immature embryos or other
organized tissue directly. In this technique, one would partially degrade
the cell walls of the chosen cells by exposing them to pectin-degrading
enzymes (pectolyases) or mechanically wound tissues in a controlled
manner.

[0046] One efficient method for delivering transforming DNA segments to
plant cells is microprojectile bombardment. In this method, particles are
coated with nucleic acids and delivered into cells by a propelling force.
Exemplary particles include those comprised of tungsten, platinum, and
preferably, gold. For the bombardment, cells in suspension are
concentrated on filters or solid culture medium. Alternatively, immature
embryos or other target cells may be arranged on solid culture medium.
The cells to be bombarded can be positioned at an appropriate distance
below the macroprojectile stopping plate. Microprojectile bombardment
techniques are widely applicable, and may be used to transform virtually
any plant species.

[0047] Agrobacterium-mediated transfer is another widely applicable system
for introducing gene loci into plant cells. An advantage of the technique
is that DNA can be introduced into whole plant tissues, thereby bypassing
the need for regeneration of an intact plant from a protoplast. Modern
Agrobacterium transformation vectors are capable of replication in E.
coli as well as Agrobacterium (and other Rhizobia), allowing for
convenient manipulations. Moreover, recent technological advances in
vectors for Agrobacterium-mediated gene transfer have improved the
arrangement of genes and restriction sites in the vectors to facilitate
the construction of vectors capable of expressing various polypeptide
coding genes. The vectors described have convenient multi-linker regions
flanked by a promoter and a polyadenylation site for direct expression of
inserted polypeptide coding genes. Additionally, Agrobacterium containing
both armed and disarmed Ti genes can be used for transformation.

[0048] In those plant strains where Agrobacterium-mediated transformation
is efficient, it is the method of choice because of the facile and
defined nature of the gene locus transfer. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA into
plant cells is well known in the art (U.S. Pat. No. 5,563,055). For
example, U.S. Pat. No. 5,349,124 describes a method of transforming plant
cells using Agrobacterium-mediated transformation. By inserting a
chimeric gene having a DNA coding sequence encoding for the full-length
B.t. toxin protein that expresses a protein toxic toward Lepidopteran
larvae, this methodology resulted in plants having resistance to such
insects.

[0049] A number of promoters have utility for plant gene expression for
any gene of interest including but not limited to selectable markers,
scorable markers, genes for pest tolerance, disease resistance,
nutritional enhancements and any other gene of agronomic interest.
Examples of constitutive promoters useful for Brassica plant gene
expression include, but are not limited to, the cauliflower mosaic virus
(CaMV) P-35S promoter, which confers constitutive, high-level expression
in most plant tissues, including monocots; a tandemly duplicated version
of the CaMV 35S promoter, the enhanced 35S promoter (P-e35S) the nopaline
synthase promoter, the octopine synthase promoter; and the figwort mosaic
virus (P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and an
enhanced version of the FMV promoter (P-eFMV) where the promoter sequence
of P-FMV is duplicated in tandem, the cauliflower mosaic virus 19S
promoter, a sugarcane bacilliform virus promoter, a commelina yellow
mottle virus promoter, and other plant DNA virus promoters known to
express in plant cells.

[0050] Exemplary nucleic acids which may be introduced to the plants of
this invention include, for example, DNA sequences or genes from another
species, or even genes or sequences which originate with or are present
in the same species, but are incorporated into recipient cells by genetic
engineering methods rather than classical reproduction or breeding
techniques. However, the term "exogenous" is also intended to refer to
genes that are not normally present in the cell being transformed, or
perhaps simply not present in the form, structure, etc., as found in the
transforming DNA segment or gene, or genes which are normally present and
that one desires to express in a manner that differs from the natural
expression pattern, e.g., to over-express. Thus, the term "exogenous"
gene or DNA is intended to refer to any gene or DNA segment that is
introduced into a recipient cell, regardless of whether a similar gene
may already be present in such a cell. The type of DNA included in the
exogenous DNA can include DNA which is already present in the plant cell,
DNA from another plant, DNA from a different organism, or a DNA generated
externally, such as a DNA sequence containing an antisense message of a
gene, or a DNA sequence encoding a synthetic or modified version of a
gene.

[0051] Many hundreds if not thousands of different genes are known and
could potentially be introduced into a Brassica plant according to the
invention. Non-limiting examples of particular genes and corresponding
phenotypes one may choose to introduce into a Brassica plant include one
or more genes for insect tolerance, such as a Bacillus thuringiensis
(B.t.) gene, pest tolerance such as genes for fungal disease control,
herbicide tolerance such as genes conferring glyphosate tolerance, and
genes for quality improvements such as yield, nutritional enhancements,
environmental or stress tolerances, or any desirable changes in plant
physiology, growth, development, morphology or plant product(s). For
example, structural genes would include any gene that confers insect
tolerance including but not limited to a Bacillus insect control protein
gene as described in WO 99/31248, herein incorporated by reference in its
entirety, U.S. Pat. No. 5,689,052, herein incorporated by reference in
its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, herein incorporated
by reference it their entirety. In another embodiment, the structural
gene can confer tolerance to the herbicide glyphosate as conferred by
genes including, but not limited to Agrobacterium strain CP4 glyphosate
resistant EPSPS gene (aroA:CP4) as described in U.S. Pat. No. 5,633,435,
herein incorporated by reference in its entirety, or glyphosate
oxidoreductase gene (GOX) as described in U.S. Pat. No. 5,463,175, herein
incorporated by reference in its entirety.

[0052] Alternatively, the DNA coding sequences can affect these phenotypes
by encoding a non-translatable RNA molecule that causes the targeted
inhibition of expression of an endogenous gene, for example via
antisense- or cosuppression-mediated mechanisms. The RNA could also be a
catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired
endogenous mRNA product. Thus, any gene which produces a protein or mRNA
which expresses a phenotype or morphology change of interest is useful
for the practice of the present invention.

D. DEFINITIONS

[0053] In the description and tables herein, a number of terms are used.
In order to provide a clear and consistent understanding of the
specification and claims, the following definitions are provided:

[0054] Allele: Any of one or more alternative forms of a gene locus, all
of which alleles relate to one trait or characteristic. In a diploid cell
or organism, the two alleles of a given gene occupy corresponding loci on
a pair of homologous chromosomes.

[0055] Backcrossing: A process in which a breeder repeatedly crosses
hybrid progeny, for example a first generation hybrid (F1), back to
one of the parents of the hybrid progeny. Backcrossing can be used to
introduce one or more single locus conversions from one genetic
background into another.

[0056] Cultivated Variety: A Brassica oleracea variety which is suitable
for consumption and meets the requirements for commercial cultivation. An
example is a broccoli variety. In addition to the plants themselves, and
the parts thereof suitable for consumption, such as the heads or leaves,
the invention comprises parts or derivatives of the plant suitable for
propagation. Examples of parts suitable for propagation are organ
tissues, such as leaves, stems, roots, shoots and the like, protoplasts,
somatic embryos, anthers, petioles, cells in culture and the like.
Derivatives suitable for propagation are for instance seeds. The plants
according to the invention can be cultivated or propagated in the
conventional manner but also by means of tissue culture techniques from
plant parts.

[0057] Crossing: The mating of two parent plants.

[0058] Cross-pollination: Fertilization by the union of two gametes from
different plants.

[0059] Diploid: A cell or organism having two sets of chromosomes.

[0060] Enzymes: Molecules which can act as catalysts in biological
reactions.

[0061] F1 Hybrid: The first generation progeny of the cross of two
nonisogenic plants.

[0062] Genotype: The genetic constitution of a cell or organism.

[0063] Haploid: A cell or organism having one set of the two sets of
chromosomes in a diploid.

[0064] Linkage: A phenomenon wherein alleles on the same chromosome tend
to segregate together more often than expected by chance if their
transmission was independent.

[0065] Marker: A readily detectable phenotype, preferably inherited in
co-dominant fashion (both alleles at a locus in a diploid heterozygote
are readily detectable), with no environmental variance component, i.e.,
heritability of 1.

[0066] Phenotype: The detectable characteristics of a cell or organism,
which characteristics are the manifestation of gene expression.

[0067] Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer
to genetic loci that control to some degree numerically representable
traits that are usually continuously distributed.

[0068] Recombination event is understood to mean a meiotic crossing-over.

[0069] Regeneration: The development of a plant from tissue culture.

[0070] Self-pollination: The transfer of pollen from the anther to the
stigma of the same plant.

[0071] Single Locus Converted (Conversion) Plant: Plants which are
developed by a plant breeding technique called backcrossing, wherein
essentially all of the desired morphological and physiological
characteristics of a broccoli variety are recovered in addition to the
characteristics of the single locus transferred into the variety via the
backcrossing technique and/or by genetic transformation.

[0072] Substantially Equivalent: A characteristic that, when compared,
does not show a statistically significant difference (e.g., p=0.05) from
the mean.

[0073] Tissue Culture: A composition comprising isolated cells of the same
or a different type or a collection of such cells organized into parts of
a plant.

[0074] Transgene: A genetic locus comprising a sequence which has been
introduced into the genome of a broccoli plant by transformation.

[0075] Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity and
understanding, it will be obvious that certain changes and modifications
may be practiced within the scope of the invention, as limited only by
the scope of the appended claims.

[0077] A deposit was made of at least 2500 seeds of broccoli hybrid RX
05991199, which comprises a reduced introgression comprising a Myb28
allele from Brassica villosa and an ELONG allele from Brassica oleracea,
as described herein. The deposit was made with the American Type Culture
Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209
USA. The deposit is assigned ATCC Accession No. PTA-13165. The date of
deposit was Aug. 24, 2012. Access to the deposits will be available
during the pendency of the application to persons entitled thereto upon
request. The deposits will be maintained in the ATCC Depository, which is
a public depository, for a period of 30 years, or 5 years after the most
recent request, or for the enforceable life of the patent, whichever is
longer, and will be replaced if nonviable during that period. Applicant
does not waive any infringement of their rights granted under this patent
or any other form of variety protection, including the Plant Variety
Protection Act (7 U.S.C. 2321 et seq.).

EXAMPLES

Example 1

Development of Parent Lines with Improved MSB Profiles

[0078] One benefit of the current invention is that parent lines may be
created comprising a reduced introgression of the invention, wherein the
lines and hybrids derived therefrom exhibit an increased proportion of
MSB/MSP and/or a greater stability of expression of MSB. The development
of such a broccoli line comprising a Myb28/ELONG reduced introgression
can be summarized as follows:

[0079] The line FT69, which has elevated levels of the phytochemical MSP
(glucoiberin) as a result of the presence of Myb28 and ELONG loci from B.
villosa (Mithen et al., 2003; Theor. Appl. Genet., 106:727-734), was
crossed with a breeding line designated BR9. The resulting progeny was
crossed with the male parent from Ironman (BRM 56-3905 SI). F1 progeny
were grown in replications in a Wageningen selection trial. From plots
designated 408, 409, 410, 411 and 412, 89 plants were selected that were
analyzed for glucoiberin (MSP) and glucoraphanin (MSB). Six plants with
the highest MSB were selected.

[0080] All 6 plants were selfed and backcrossed with the recurrent parent
BRM 56-3905 SI. The 6 selfings and 6 BC1 were planted in replicated
selection trials in Wageningen. Each BC1 (where BC=backcross) had 2 plots
with 24 plants/plot. From these 12 plots, a total of 84 plants were
selected that most resembled the recurrent parent. Heads from these
selections were sent for MSP and MSB analyses. Seven plants with highest
MSB were kept for further selfing and BC. All 7 BC1 plants were selfed
and backcrossed with the recurrent parent (BRM 56-3905 SI). Selfings and
5 BC2 populations were sown and selected in replicated Wageningen Autumn
selection trials. A total of 73 plants that resembled the recurrent
parent most were selected and sent for MSP/MSB analyses. Eight BC2 plants
with highest MSB levels were kept for further selfing and BC.

[0081] All 8 BC2 plants were selfed and backcrossed with the recurrent
parent (BRM 56-3905 SI). Four BC3 were obtained and planted in 1-4
replications in Wageningen. 47 plants closest to the recurrent parent
were sent for MSP/MSB analyses. 7 BC3 plants with highest MSB levels were
kept for further selfing and BC. All 7 BC3 plants were selfed and
backcrossed with the recurrent parent (BRM 56-3905 SI). 6 BC4 were
obtained and these were planted in selection trials. 54 plants closest to
the recurrent parent were sent for MSP/MSB analyses. 8 BC4 plants with
the highest MSB levels were kept for further selfing and BC.

[0082] All 8 BC4 plants were selfed. 7 BC4 plants produced selfed seed and
the BC4F2 were planted in unreplicated Wageningen selection trials. 37
BC4F2 plants closest to the recurrent parent were selected and these were
sent for MSP/MSB analyses. 4 BC4F2 plants with highest MSB levels were
kept for further selfing and BC.

[0083] All 4 BC4F2 plants were selfed. All 4 produced selfed seed and the
4 BC4F3 were planted in unreplicated selection trials. 12 BC4F3 plants
closest to the recurrent parent were selected and sent for MSP/MSB
analyses. 4 BC4F2 plants with the highest MSB levels were kept for
further selfing and BC. All selected plants from plot 1169 (570114) had
equally high MSB level of average 2.3 mmol/kg, or about 5× the
level of the standard reference variety (General). This indicated that
the source has alleles fixed for high MSB.

[0084] All 3 selections from 1169 (BC4F3) were selfed. All 3 produced seed
and these were planted in selection trials. SNP13-580333 (1169-1, A75-1)
was the most uniform on type and 5 plants were selected for MSB check.
All 5 had equally high levels of MSB of about 3.2 mmol/kg. 1169-1, also
designated BRM 53-3934 SI, was put on tissue culture for increase and use
as a male parent to RX-1199 (RX 05991199). An analysis of BRM 53-3934 SI
revealed that it contained the Myb28 reduced introgression, which results
in a parent line with the ability to consistently produce progeny with
glucosinolates comprising an increased proportion of MSB/MSP relative to
plants having the B. villosa ELONG locus, as shown below.

Example 2

Analysis of Hybrid Broccoli Lines Having Elevated Glucoraphanin

[0085] Self and outcrosses were made using different standard broccoli
lines as female parents as shown in Table 1 in crosses with the
following: (1) FT69 (high glucoiberin line with ELONG and Myb28 from B.
villosa), (2) SNP13-580333 (high glucoraphanin line with ELONG from B.
oleracea and Myb28 from B. villosa, as described above), and (3)
SNP88-BRM51-1210 (high glucoiberin line ELONG and Myb28 from B. villosa).
As can be seen, regular broccoli lines have a relatively low amount of
total glucosinolates. The ratio between MSB and MSP is particularly shown
to be depending on the regular broccoli female line in the case of FT69
(428-11-69), which comprises ELONG and Myb28 alleles from B. villosa. In
contrast, in all the crosses with SNP13-580333 most of the glucosinolates
are glucoraphanin. This indicates the benefit of the reduced
introgression in consistently producing hybrid varieties comprising
elevated glucoraphanin in crosses with multiple different second parents.

[0086] As explained above, one embodiment of the invention comprises
producing broccoli hybrids wherein one or both parents of the hybrid
comprise a Myb28 reduced introgression and, as a result, exhibit an
elevated MSB content and/or more stable MSB content relative to hybrids
lacking a parent comprising the introgression. One example of such a
hybrid that was produced is the hybrid RX 05991199. This hybrid was
created by crossing parents BRM 53-3934 SI and BRM 56-3907 CMS, typically
with BRM 53-3934 SI as a male parent. The production of BRM 53-3934 SI is
described in Example 1 above. As explained, this parent contains the
Myb28 reduced introgression. The female parent BRM 56-3907 CMS is a known
inbred that served as the parent of the commercial hybrid "Ironman." BRM
56-3907 CMS is the subject of, and is described in, EU Plant Variety
Rights Certificate #20341, granted Jun. 18, 2007.

[0087] A description of the physiological and morphological
characteristics of broccoli hybrid RX 05991199 and the parent lines
thereof is presented below.

[0090] Genetic marker assays were developed to genotype for Myb28 and
ELONG alleles. The assays thus permit identification of a reduced
introgression in accordance with the invention as well as marker assisted
introduction of the reduced introgression into any other genotype.

[0091] A. Markers for Detection of ELONG

[0092] A marker designated QTL1-BoGLS-ELONG was developed and permits
detection of the presence or absence of a B. villosa ELONG allele. This
marker can be detected using the primer pair AF399834F2:
5'-cggattttcaaattttctcg-3' (SEQ ID NO:1) and AF399834R2:
5'-atttcgcatgaccactaggc-3' (SEQ ID NO:2). To detect the marker, plates
were loaded with 20 ng DNA template (sample) in a 2 μL volume. 34,
master mix (0.437 μL water, 2.5 μL Q PCR(ROX) mix, 0.063 uL assay
mix) was added to each well for a final volume of 5 μL. PCR conditions
were as follows: 95° C. for 15 min then 40 cycles of 95° C.
for 15 sec, 60° C. for 1 min. FIG. 4 shows results of an assay for
different ELONG alleles using the marker. The V allele is indicative of
the B. villosa ELONG allele, while A, B and C alleles are examples of
alleles found in broccoli without a B. villosa ELONG allele.

[0093] B. Marker for the Detection of Myb28

[0094] The identification of genetic polymorphisms in Myb28 alleles and
their use as genetic markers is described in U.S. Provisional Appln. Ser.
No. 61/700,731, filed concurrently herewith, the disclosure of which is
incorporated herein by reference in its entirety. Specifically, sequence
alignments were described therein between B. oleracea alleles and the
corresponding Myb28 sequences to identify polymorphisms that can be used
for marker based selection for a Myb28 allele of choice. The results are
shown in FIG. 5, which is an alignment between a consensus sequence of
the Myb28 locus from B. villosa contained in broccoli variety FT69, and a
consensus sequence of the corresponding locus from broccoli without
increased level of glucosinolate, e.g. B. oleracea, (Oleracea). Shown are
26 polymorphisms (e.g. single feature polymorphisms (SFPs)--of which
there are 16 SNPs and 10 indels) detected in a sequence with a total
length of 2202 bp. Any of these or other identified polymorphisms may be
used as genetic markers for the presence or absence of a desired Myb28,
including from B. oleracea or B. villosa.

[0095] A TaqMan assay (NBOLI009111370) was designed based on one of the
sequence polymorphisms identified, as follows:

[0096] All of the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of the
present disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be applied to
the compositions and/or methods and in the steps or in the sequence of
steps of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be apparent
that certain agents that are both chemically and physiologically related
may be substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by the
appended claims.